Project Summary/Abstract This competitive renewal R01 application is focused on defining the molecular mechanisms controlling human adrenarche. Adrenarche denotes the marked increase in adrenal DHEA-S production that normally occurs at around 6 years of age and initiates the prepubertal development of axillary and pubic hair. Premature adrenarche may be an early indicator of diseases that manifest in adulthood, which increases the clinical relevance for understanding the mechanisms controlling adrenarche. While the exact trigger that induces adrenarche remains poorly understood, we have made significant progress in defining adrenal alterations that occur during adrenarche. Most notable is the development/expansion of the adrenal zona reticularis with expression of the key enzymes needed to produce DHEA-S. While DHEA-S has been an appropriate marker for progression of adrenarche, it has no androgen activity in itself, and requires peripheral conversation to more active compounds. In this application, we propose that adrenarche and particularly pubarche result from adrenal production of bioactive androgens. In this proposal, we focus on the definition of active androgens produced by the adrenal, the biosynthetic pathways for these steroids and the intra-adrenal molecular mechanisms controlling the androgen production. Specific Aim 1 will define the steroid metabolome of normal and premature adrenarche. Our preliminary findings suggest that adrenarche and premature adrenarche are associated with increases in a broad group of steroids including bioactive androgens. Our recent advances in the use of LC-MS/MS for steroid hormone analysis provides the opportunity for us to examine 30 steroids during adrenarche. Studies proposed in Specific Aim 2 will characterize the adrenal biosynthetic pathways for testosterone and the novel bioactive androgens 11-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT). Our past work determined that many adrenal steroid-metabolizing enzymes are expressed in a tight adrenal cortex zone-specific manner. Here, we propose that production of adrenal active androgen is facilitated by cooperation between the zona reticularis and glucocorticoid-producing zona fasciculata. The proposed research will characterize this unique ?two-zone? steroidogenic pathway. In Specific Aim 3, we will define the molecular mechanisms regulating adrenal zona reticularis expression of the enzymes required for bioactive androgen synthesis. Our preliminary findings suggest a key role for methylation in the regulation of zona reticularis capacity to make androgens. Collectively, the proposed studies will cause a shift in our concepts regarding the adrenal as an endocrine organ and particularly in its contribution to circulating androgens. In addition, the study will define the role of the adrenal-specific steroids, 11OHT and 11KT as potential regulators of the androgen receptor and thus the androgenic manifestations of adrenarche. Successful completion of the proposed studies also has the potential of impacting clinical care of children and women with disorders of androgen excess.